Serotyping of Streptococcus pneumoniae isolated from Tehran by Multiplex PCR: Are serotypes of clinical and carrier isolates identical?

Background and Objectives Streptococcus pneumoniae is the most common cause of invasive infections among both young children and elderly people. Common serotypes causing invasive diseases and the emergence of carriers of Streptococcus pneumoniae in Iran is not yet known. Past-vaccine surveillance studies of serotype prevalence patterns in Iran are necessary to monitor the epidemiology of Streptococcus pneumoniae. Because of variation of pneumococcal serotypes in different geographical regions, in this study we evaluated common serotypes causing pneumococcal infections and healthy carrier children in Tehran by Multiplex PCR. Materials and Methods A total of 150 nasopharyngeal swabs were collected from healthy children in Tehran between December 2011 and August a2012, and 100 clinical samples were collected. Identification was performed by biochemical and molecular tests. Serotyping was done by multiplex PCR. We designed primers based on the sequences available for the routine capsular types and combined them into six multiplex PCR. Results From 150 nasopharyngeal swabs, 40 isolates of Streptococcus pneumoniae were identified after identification tests. Thirty six clinical isolates were also detected among clinical samples. Four serotypes (19A, 6, 3, 23F) of S. pneumoniae accounted for 55.7% of both sets of strains isolated from nasal carriage and clinical samples. Serotype 19A was the most common serotype among both groups. Conclusion The multiplex PCR approach was successfully adapted to identify serotypes from more than 91% of the isolates tested. Among S. pneumoniae isolates in Tehran, the most prevalent serotypes were similar among carriage and invasive isolates. Continued monitoring of common serotypes of Streptococcus pneumoniae is essential for future vaccine formulation in Iran.


INTRODUCTION
Streptococcus pneumoniae is an important infectious agent causing of life-threatening infection diseases worldwide, particularly in children and the elderly people (1). World Health Organization (WHO) has reported that nearly 1 million children die of pneumococcal disease annually, mostly in developing countries (2). Several diseases are caused by S. pneumoniae including sepsis, pneumonia, meningitis, otitis media, sinusitis and bronchitis (2, 3). Antimicrobial resistance in S. pneumoniae has increased over the last three decades (4). The antibiotic-resistant strains are often associated with multiple serotypes and the rates of antibiotic resistance varies geographically (4, 5). The increase of antibiotic resistance has complicated treatment of pneumococcal infections (6).
S. pneumoniae can produce at least 90 capsular serotypes (7), but only a few of these cause most cases of invasive diseases (8). Identification of these serotypes is essential for production of the new generation of conjugate vaccines and treatment protocols (5). There are geographical differences in prevalence and distribution of serotypes (9). Hence, it is necessary to determine the prevalent serotypes of S. pneumoniae in different geographical areas. Pneumococcal polysaccharide vaccines and pneumococcal conjugate vaccines are two types of vaccines that are used in control of invasive diseases. Polysaccharide vaccines are not effective against otitis media or nasopharyngeal carriage and are poorly immunogenic in children less than age 2 years (10, 11). Pneumococcal conjugate vaccine 7-valent (PCV7) was routinely used in USA and Europe, but it has not been used since March 2010, and vaccination with PCV13 has started in many countries since 2010 (6). Use of an effective conjugate vaccine during infancy reduced the rates of invasive pneumococcal infections and antibiotic resistance associated with serotypes (12). However, infections caused by other serotypes have increased (4). Although previous studies showed that distribution of pneumococcal diseases and the rates of antimicrobial resistance in Iran are high (13,14), a few studies have been performed on the serotypes distribution of S.pneumoniae in Iran.
Traditionally, the Quellung reaction is used for serotyping of S. pneumoniae. However, the high cost and require skilled technicians have stimulated the need for the development of PCR-based serotyping systems (15)(16)(17). Molecular methods such as multiplex PCR are faster and simpler as well as have higher sensitivity and lower cost than the cultural methods. Furthermore, Multiplex PCR can be used directly on clinical samples obtained from culturenegative patients (18). The aim of this study was to determine the prevalent pneumococcal serotypes and to compare serotypes in healthy carrier children and clinical samples of S. pneumoniae by Multiplex-PCR method.

MATeRIALS AND MeTHODS
Bacterial isolates. In a cross-sectional study, 150 nasopharyngeal swabs were collected from healthy children less than 5 years of age having referred to 4 main care centers (Torkamani, Shobeir, Hazrate Roghayyeh, Ameneh) and Children's Medical Center of Tehran from December 2011 to August 2012. Nasopharyngeal swabs were kept in Stuart transport medium (Oxoid Limited, Hampshire, England) which were sent to the Department of Bacteriology in Pasteur Institute of Iran. Nasopharyngeal swabs were inoculated onto 5% sheep blood agar and incubated overnight at 37°C in 5% CO 2 . The colonies that were morphologically similar to S. pneumoniae were subcultured onto 5% sheep blood agar. One hundred clinical samples including blood, cerebrospinal and pleural fluids were collected from patients of Imam Khomeini, Sina, Shohada and Hazrate Rasoul hospitals during September 2008 -March 2010. The clinical samples were obtained from invasive and non-invasive infection of both pediatric and adult patients.

Phenotypic and Molecular Identification
Methods. Identification of S. pneumoniae isolates were performed by biochemical methods such as Gram staining, colony morphology (alpha-hemolytic, small, gray, and showing mucoid colonies), optochin susceptibility, and bile solubility. Optochin disks (6 mm; MAST diagnostics, Bostle, Mersey side) were applied to 5% sheep blood agar. After overnight incubation at 37°C in 5% CO 2 atmosphere, isolates displaying zones ≥14 mm in diameter were considered as S. pneumoniae. Complete clearing of heavy bacterial suspensions (1 ml) matching the 2.0 McFarland turbidity in the deoxycholate tube after incubation at room temperature for 15 min was indicative of S. pneumoniae. Molecular tests were used for confirmation of identification of isolates by PCR for cpsA gene as described previously (19). S. pneumoniae ATCC 6305 was used as control strain in all of identification tests.
Preparation of chromosal DNA. Genomic DNAs were extracted using a DNA extraction Kit (Metabion™ international AG, Germany) according to the manufacture's protocol. Finally, the harvested DNA pellet was re-suspended in RNase-Tris-EDTA to provide 70 µl of DNA sample.

Identification of Streptococcus pneumoniae
isolates. From 150 nasopharyngeal swabs, 40 isolates were isolated and identified as S. pneumoniae using standard tests. In addition, 36 clinical isolates were identified among patients. All pneumococcal strains were optochin susceptible and bile soluble. Presence of cpsA gene was confirmed in all 76 S. pneumoniae isolates (Fig. 1). Overall prevalence of nasal carriage among children was 26.6%.

Multiplex PCRs and Serotypes distribution.
Of the 76 isolates of S. pneumoniae, 70 isolates (92.1%) were serotyped with the available specific primers by multiplex PCR (Fig. 2). Among S. pneumoniae strains isolated from nasal carriage, 37 (92.5%) were serotyped successfully by multiplex PCR. Similarly, multiplex PCR was successful in serotyping of 33 of 36 clinical isolates of S. pneumoniae (91.6%). All results were consistent with the scheme containing classification of serotypes in Table 1. Primers concentration in each multiplex PCR reaction was variable from 0.5 µM to 1.5 µM to obtain the best result.

DISCUSSION
Streptococcus pneumoniae is a common cause of respiratory infections requiring hospitalization in young children worldwide with increasing rates of antibiotic resistance (21). Generally, serotype determination of S. pneumoniae is performed by culture of the organism followed by serological detection of the capsular type by quellung test. This method of serotyping is not widely feasible due to the high cost of antisera, difficulties in interpretation, and requirement of technical expertise (20). The development of PCR-based serotyping has the       potential to overcome some of the difficulties associated with the conventional serologic method. In this survey, multiplex PCR was successful in serotyping of S. pneumoniae isolates. However, 6 isolates (7.8%) could not be typed by multiplex PCR test. These isolates could be non-serotypeable, but it is important to note that multiplex PCR system used in this study was designed to only detect 20 common serotypes which were previously reported (20,(22)(23)(24). As expected, the first three reactions in our multiplex PCR scheme detected the major previously reported serotypes from different countries. Interestingly, among the 76 isolated S. pneumoniae strains in this study, 83.8% were serotyped following three reactions of multiplex PCR indicating high sensitivity of this method in serotyping of S. pneumoniae strains. Similar to our findings, Pai et al (20) showed that these three reactions were able to detect more than 60% of the strains in the United States. Furthermore, using the first three reactions of this scheme in the Latin America, 89.1% of S. pneumoniae isolates were accurately serotyped (22). These results show that multiplex PCR can reduce the costs of serotyping compared to the conventional serotyping methods.
In this study, serotype distribution was examined in both carriers and clinical isolates. MP-PCR allowed serotyping in 37 (92.5%) of 40 nasopharyngeal carrier of S. pneumoniae in healthy children less than five years-old, and 33 (91.6%) of 36 with invasive pneumococcal disease (IPD). Both groups showed 13 serotypes of 20 serotypes examined. These serotypes were similar in both groups indicating the importance of nasopharyngeal colonization in the development of serious pneumococcal infections. However, there were differences in the position of some serotypes in the two groups that may be due to low number of specimens. To determine the actual position of serotypes, serotyping should be done on more specimens.
The serotype distribution in this study is not similar to that reported in previous study of Iran (14). The differences may be due to the use of different methods for serotyping. In the previous study, serotyping had been performed using antisera.
Among S. pneumoniae isolates in Tehran, Five serotypes were included about 60% of carriage and invasive isolates. Similarly, In Brazil, USA and UK a few pneumococcal serotypes has been related to the majority of nasopharyngeal carriage and invasive disease (24-26), In contrast, in Asia a wide variety of serotypes has been related to nasopharyngeal carriage and invasive disease (27,28). In the present study, serotypes 19A and 6 were the most common serotypes in carriers and clinical isolates. High prevalence of serotype 3 was shown among carrier isolates, whereas it was rarely found in clinical isolates. Overall, the serotypes detected among the nasopharynx and serotypes causing invasive disease often are represented in the PCV13. Moreover, results showed that 48.5% of the pneumococcal serotypes were from serotypes of pneumococcal conjugate 7-valent vaccine (4, 6B, 9V, 14, 18C, 19F, 23F). However, Serotype 18C was not found among S. pneumoniae isolates.
Previous studies indicate that young children in Iran experience a high prevalence of S. pneumoniae carriage rate. A study of 1300 healthy children less than ten years-old in Tehran, Iran showed pneumococcal nasopharyngeal carriage rate of 44.1% (14). Another study showed a lower nasopharyngeal carriage rate (15.7%) for S. pneumoniae in Iranian healthy adolescents that decreased with age (13). Despite the high prevalence of carriage in Iranian children, so far vaccination has not been carried out in this country. Therefore, continued monitoring of common serotypes of S. pneumoniae is essential for future vaccine formulation in Iran. Since the serotypes distribution are similar among both nasopharyngeal carrier and clinical isolates in Tehran, epidemiological studies to investigate the prevalent serotypes in nasopharyngeal carriers could aid the development of a suitable vaccine for prevention of invasive pneumococcal diseases.